The present invention relates generally to solid phase methods for conducting specific binding assays and more specifically to the use of chromatographic techniques in conducting nucleic acid hybridization assays.
The use of specific binding assays has been found to be of great value in a variety of clinical applications. Various biological fluids and tissue samples can be analyzed for a wide variety of components such as drugs, hormones, enzymes, proteins, antibodies, DNA and RNA fragments and other biological material.
Specific binding assays include those assays wherein an analyte is measured which is a member of a specific binding pair consisting of a ligand and a receptor. The ligand and the receptor are related in that the receptor specifically binds to the ligand, being capable of distinguishing the ligand from other sample constituents having similar characteristics. Immunological assays depend on reactions between immunoglobulins (antibodies) which are capable of binding with specific antigenic determinants of various compounds and materials (antigens). Specific binding assays may also involve nucleic acid hybridization reactions wherein single strands of oligo or polynucleotides (e.g. DNA or RNA) hybridize through hydrogen bond formation with strands of other polynucleotides comprising complementary base sequences. Still other specific binding assays are known, such as those involving hormone receptors and biotin avidin, which involve neither immunological reactions nor hybridization.
Various types of specific binding assay techniques are also known for the detection of specific nucleic acid sequences. Such assays utilize nucleic acid hybridization procedures wherein complementary polynucleotide sequences of single stranded nucleic acid polymers recognize each other (nucleate) and interact (anneal) to form a stable duplex structure.
Nucleic acid hybridizations have been carried out in both homogeneous (in-solution phase) media and heterogeneous (solution/solid phase) media. See, for example, EP A-288 737, W086/07387 and U.S. Pat. No. 4,787,963. In homogeneous media, the hybridized polynucleotide sequences typically are separated from the unhybridized sequences by passing them over a hydroxyapatite column under appropriate conditions. Separation can also be achieved by gel exclusion as taught in Kuhns, EP A-278,220; by polycationic binding supports as taught in EP-A-281-390; or by precipitating agents as taught in EP-A-167,366. Several references teach heterogeneous hybridizations, including a sandwich hybridization taught by Ranki, et al., U.S. Pat. No. 4,563,419.
Despite the great advances that have been made with respect to specific binding assay techniques in recent years, there still remain significant opportunities for improvement of these techniques, particularly where nucleic acid hybridizations are concerned. Current solution phase hybridization procedures require significant incubation times, typically on the order of several hours, to permit diffusion and nucleation of complementary sequences. Even under known methods for acceleration of hybridization, incubations require about 1-2 hours. Then a separation step is required to separate the duplexes. Solid phase hybridizations make separation easier but often involve increased incubation times. Since only one of the two complementary nucleic acids is able to diffuse freely, diffusion and nucleation does not occur as rapidly at the surface of the solid phase.
MacConnell, U.S. Pat. No. 4,787,963 discloses a method and apparatus for improving the kinetics of heterogeneous hybridizations. By electrophoretically concentrating nucleic acid probes at a membrane to which the target sequences are bound, the chances of nucleation and annealing are increased. The reference claims hybridization occurs in a matter of 15-30 minutes, rather than hours.
EP A 0 288 737 discloses another method for achieving more rapid heterogeneous hybridizations. The time required for diffusion of sequences to the internal pores of a nitrocellulose membrane to which probe is bound necessitates longer incubation times. Therefore, the reference discloses a solid phase comprising water suspensible microparticles to which probe is bound. The decreased diffusion distances permit nucleation in about 30 minutes, and the separation of microparticles is easily accomplished by filtration.
Nevertheless, new systems involving solid phase assay devices having improved kinetics are highly desired. Such devices would preferably be simple to use in conducting assays for a wide variety of nucleic acids and would be capable of providing for hybridization in less than 15-30 minutes.